Publications Using the CellRaft® Technology
Sirtuin 6 (SIRT6) is a deacylase and mono-ADP-ribosyltransferase enzyme implicated in multiple aging-related pathways including DNA repair, telomere maintenance, and metabolism regulation. A strong correlation between SIRT6 activity and longevity has been observed across species, and genetic polymorphisms in the SIRT6 gene correlate with human longevity in SNP analyses. To demonstrate that the centSIRT6 allele improves DNA repair efficiency, the authors used the CellRaft AIR system to isolate monoclonal mesenchymal stem cells (MSCs) with a centSIRT6 knock-in. MSCs with the centSIRT6 allele demonstrated a higher efficiency of DNA repair and increased survival upon treatment with methyl methanesul-fonate, a potent DNA-damaging agent, supporting the hypothesis that the centSIRT6 allele contributes to human longevity by improving genome maintenance.
The authors present a novel methodology to address the artifacts associated with cell lysis and whole genome amplification during genome-wide DNA mutation analysis, termed Single-Cell Multiple Displacement Amplification (SCMDA). SCMDA and SCcaller were validated by direct comparison of SNVs from amplified single cells and unamplified clones derived from cells in the same population of early passage human primary fibroblasts. The CellRaft® Technology was utilized for multiple steps in the validation workflow, including isolation of single cells and generation of single cell clones. Single cells were plated on CytoSort™ Arrays, isolated, and subjected to downstream SCMDA, library preparation, and sequencing. Empty CellRafts® were also isolated to serve as negative controls in WGA. To generate single cell clones, cells were plated on CytoSort™ Arrays and isolated once the clones reached confluency on the CellRaft®, approximately 10-16 cells per raft, for further expansion in 96-well plates. Lastly, to generate kindred single cells and clones, small clones were transferred from 96-well and seeded on a fresh CellRaft Arrayto isolate single cells. This method, and the corresponding single-cell variant caller SCcaller, provide a foundation for standardizing somatic mutation analysis in single-cell genomics.
It has long been known that genetic material is mutable at a rate subject to natural selection, but multicellular organisms have a somatic genome with a mutation rate that differs from the germline mutation rate. However, a lack of reliable methods to measure somatic mutation frequencies in DNA have precluded a direct comparison in mutation rates between somatic and germline cells. Vijg and colleagues present the first direct comparison of mutation rates in human and mouse single somatic cells, both of which are further compared to human and mouse de novo germline mutation rates. The CellRaft® Technology is utilized here to isolate single cells for downstream single cell whole genome sequencing after amplification. The results presented suggest that somatic mutation frequencies are significantly higher than germline mutation frequencies, which may point toward somatic mutations as a conserved mechanism of aging.
Neocortical neurons are among the most diverse and longest-lived mammalian cells, and human-specific brain phenotypes are attributed to neocortical expansion during evolution. McConnell and coworkers assembled a brain copy number variation (CNV) atlas to reveal the frequency of neocortical neurons with complex karyotypes and the associated variability among individuals. These CNVs represent rare variants with strong contributions to genetic risks for schizophrenia, autism, and other neurological disorders. The CellRaft® Technology was used to isolate, and verify the integrity of, single nuclei following flow sorting to assess the quality of the whole genome amplification (WGA) method utilized. The authors provide evidence that a functional consequence of CNV neurons may be selective vulnerability to aging-associated atrophy.
Cohesin regulates three-dimensional gene organization and gene expression contributing an important role in the structure integrity of many proteins. PDS5A and PDS5B are two subunits that make up HEAT repeat proteins that interact with cohesin proteins. To understand more about the role these two subunits play in cohesin and gene expression, two stable knockout lines were constructed using the CellRaft AIR System. Cells were transfected with two plasmids, allowed to settle for a day and then seeded onto a CellRaft Array to expand. Once clones were generated and measured with fluorescence markers to determine successful edit, CellRafts containing colonies of interest were isolated using the CellRaft AIR System. Downstream, cells were sequenced and analyzed for further investigation against the role the two subunits play in gene expression when involved with the cohesin proteins.
A major limitation with pooled screening platforms is the inability to identify phenotypic characteristics associated with cells of interest. Most platforms generate simple readouts such as viability and fluorescence. Using a method termed “Raft-Seq” this paper demonstrates how the CellRaft AIR System was able to distinguish pathogenic point mutations of the mitochondrial regulator Mitofusion 2. The CellRaft AIR System was used to generate clones for monitoring phenotypes as well as isolating cells of interest for downstream sequencing. The CellRaft Technology was compared against 4 other cell screening-imaging platforms (Arrayed screening, In situ sequencing, photo-activation, and CRaft-ID) and among 7 comparison criteria (Imaging constraints, how cells are collected, genotype resolution, live cell collection, genotyping method, pooled scalability, and bottleneck). Overall, the platforms and comparison criteria, the CellRaft Technology dominated the competition by making it possible to examine phenotypic characteristics of live cells on a single cell genotype resolution with an ability to isolate out cells of interest for further downstream NGS.
Identifying genetic and genomic characteristics that determine the functional or phenotypic properties of individual tissues and cells in multicellular organisms is a fundamental problem in modern biology. While high-throughput techniques, such as RNA-seq, are indispensable tools, they are most often performed on bulk tissue samples and inherently blur the properties of individual cells within a heterogeneous tissue. The authors present the CellRaft® Technology as a method that provides two key advantages over existing single cell isolation approaches for genomics: selective isolation without the requirement for a pre-selection step, and the ability to sort cells based on complex temporal or spatial phenotypes or cellular markers. To evaluate this approach, CFPAC-1 cells were plated on CytoSort™ Arrays and treated with 0, 2, or 5 nM gemcitabine and allowed to grow on the array for 4 days, with strongly restricted growth seen in the cells treated with 5 nM gemcitabine. Cells of interest, either proliferative or non-proliferative, were subsequently isolated and sequenced, thereby linking a proliferative pancreatic cancer cell phenotype, in the presence of a chemotherapeutic agent, to the altered transcriptome of the cells.
Mutations are the driving molecular causes of various biological processes such as developmental and cancer. Recent findings of genomic heterogeneity among ostensibly homogeneous cell populations such as cancer cells demand genomic characterization of mutations at the individual cell level to better understand the underlying biology. Additionally, single cell technologies make genomic analysis feasible for the characterization of rare cells such as circulating tumor cells. Due to its high sensitivity, next generation sequencing (NGS) represents the ideal technology to analyze a collection of mutations in single cells. The challenge, however, is that single cells yield limited amounts of DNA, that need to be amplified prior to NGS. To overcome this challenge, whole genome amplification (WGA), coupled with multiplex PCR-based targeted enrichment, were tested for mutation detection in single cells isolated from two colon cancer cell lines, Lovo and HT29.
Genomic mosaicism is prevalent throughout human somatic tissues and is much more common than previously thought. Here, we describe step-by-step methods to isolate neuronal nuclei from human brain and identify megabase-scale copy number variants (CNVs) in single nuclei. The approach detailed herein includes use of CellRaft technology for single-nucleus isolation, the PicoPLEX approach to whole-genome amplification and library preparation, and a pooled library purification protocol, termed Gel2Gel, which has been developed in our laboratory. These methods are focused toward neuroscience research, but are adaptable to many biomedical fields.
In order to establish a causal relationship between somatic mutations and aging, mutational events must be directly identified in primary human tissues. Single cell sequencing holds promise for the detection of a full complement of mutations in somatic cells, overcoming the challenges that arise due to the random nature and very low abundance of most somatic mutations; however, the genome amplification procedures required for single cell genomics have a high error rate. To address this problem, Vijg, et al. developed a highly accurate single cell multiple displacement amplification (SCMDA) to comprehensively determine the full spectrum of base substitutions in a single somatic cell, and thereby assess mutation accumulation as a function of age in human B lymphocytes from healthy individuals. To aid in this analysis, bulk B lymphocytes were isolated from PBMCs and subsequently plated on gelatin coated CytoSort™ Arrays. Using the CellRaft® Technology, single B lymphocytes were isolated and collected in PCR tubes for SCMDA and downstream analysis of somatic mutations.
